Renzo Bazzo

Structural Analysis of the Epitope of the Anti-HIV Antibody 2F5 Sheds Light into Its Mechanism of Neutralization and HIV Fusion

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I beta turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.

Contribution of proline-14 to the structure and actions of melittin

The structure and dynamic properties of bee venom molittin and a synthetic analogue. [Ala14]‐melittin (melittin P14A), are compared, using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy and amide exchange measurements in methanol. P14A is shown to adopt a regular, stable α‐helical conformation in solution without the flexibility around the Pro‐14 residue found in melittin. P14A has twice the hemolytic activity of melittin but is less able to induce voltage‐dependent ion conductance in planar bilayers. The results indicate that helix flexibility afforded by the Pro‐14 residue promotes the ability of melittin to adopt the transbilayer associates thought to underlie ion translocation.

A theoretical analysis of homonuclear cross-polarization coherence transfer in liquids

Abstract A theoretical analysis of homonuclear rotating-frame coherence transfer in liquids is presented. A general procedure is developed to describe coherence transfer under the influence of a phase-modulated RF field. The mechanism of coherence transfer is discussed and compared with other two-dimensional homonuclear correlation techniques.

Primary sequence dependence of conformation in oligomannose oligosaccharides

The oligomannose series of oligosaccharides from bovine thyroglobulin (BTG) and the variant surface glycoprotein (VSG) ofTrypanosoma brucei have been isolated and sequenced by1H NMR. The structure of Man9GlcNAc2, the parent molecule of the series, is shown below. Structural isomerism occurs within this series through the removal of residues D1, D2, D3, and C. Using spin-spin coupling and chemical shift data the rotamer distributions about the dihedral angle ω for the Manα1-6Man\ and Manα1-6Manα linkages were determined for each member of the series. It is shown that the dihedral angle ω of the Manα1-6Man\ linkage exhibits low flexibility with a preference for the ω = 180° conformation when residue D2 is present and high flexibility when this residue is absent. Flexibility of ω for the Manα1-6Manα is largely independent of primary sequence and is intermediate between the two Manoα1-6Man\ extremes, again with a preference for the ω = 180° conformation. There are, however, data which indicate that removal of residue D3 may confer additional flexibility upon the dihedral angle ω of the Manα1-6Manα linkage. Molecular graphics modelling, together with chemical and enzymatic modification studies, suggest that the origin of the observed primary sequence dependence of the Manα1-6Man\ linkage arises from steric factors. On the basis of these observations taken together with previous work, it is postulated that recognition of individual oligomannose conformations may play a role in the control of N-linked oligosaccharide biosynthesis.

Uncertainties in structural determinations of oligosaccharide conformation, using measurements of nuclear Overhauser effects

A fundamental problem in the determination of molecular structure by n.m.r. spectroscopy is insufficient experimental constraints. This problem is particularly marked for oligosaccharides, where few constraints are available across glycosidic linkages. By calculating distances as a function of dihedral angle, it is shown that, in general, two n.O.e. constraints result in two possible conformations for each glycosidic linkage, one of which can usually be discarded on the basis of model building or energy calculations. Using these calculations, an estimate of the uncertainty in the structure can be obtained.

Full simulation of ROESY, including the Hartmann-Hahn effects

Abstract A general method for the computation of cross peak intensities in ROESY (rotating frame NOE spectroscopy) in illustrated in this paper. A suitable formalism, based on the density matrix theory, enables all the magnetization transfer processes occurring in ROESY, both coherent and incoherent, to be treated in a unified fashion. A Fortran computer program has been used to perform numerical simulations. This treatment aims at extending the range of applicability of ROESY, particularly in the conformational studies of organic and biological molecules by NMR.

Improved sensitivity in indirect monitoring of chemical shifts of proton-heteronuclear spin pairs (1H-13C and 1H-15N) in 3D and 4D NMR spectroscopy

In three-dimensional and four-dimensional experiments on doubly labelled proteins not only heteronuclear (13C or 15N) but also proton (1H) frequencies are often indirectly monitored, rather than being directly observed. In this communication we show how in these experiments by overlaying 1H and heteronuclear evolutions one can obtain decreased apparent relaxation rates of 1H signals, yielding improved sensitivity. The new method applies to spin pairs like 1H-15N, as in amide groups, or 1H-13C, as in methine groups of alpha or aromatic systems.

Prime Site Binding Inhibitors of the Hepatitis C Virus NS3/4A Serine Protease

Infection by hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer. Much effort for a therapy is currently devoted to the search of inhibitors of the serine protease NS3/4A, which is required for maturation of viral polyprotein.